Transfer of the epicardial-cardiac organotypic culture model to support the ex vivo screening of gene therapy candidates
Lead Research Organisation:
University of Surrey
Department Name: Biochemistry & Physiology
Abstract
The recovery capacity of the adult heart after injury is severely limited by the low number of regenerating cells within this tissue. The epicardium, the most external layer of the heart, contains cells able to home to the injured heart muscle and promote its recovery. We developed an innovative model based on thin slices obtained from leftover pig heart tissues that mimics in a dish the behaviour of the epicardium. In this project, we will teach other laboratories working on the epicardium how to prepare and use our model in their own research.
The adoption of our model by our partners will provide an alternative strategy to test their innovative gene therapy ideas, reducing their use of regulated procedures (surgeries) performed on animals.
In addition, we will present our model to several other interested laboratories and industrial partners working on similar science projects in order to encourage further uptake and further reduction of animal use.
The adoption of our model by our partners will provide an alternative strategy to test their innovative gene therapy ideas, reducing their use of regulated procedures (surgeries) performed on animals.
In addition, we will present our model to several other interested laboratories and industrial partners working on similar science projects in order to encourage further uptake and further reduction of animal use.
Technical Summary
The emerging role of the epicardium in the heart regeneration points at a largely unexplored reparative potential, boosting the number of studies focusing on this tissue and increasing the number of animals used for this research. Most studies are performed in simplistic single cell culture or small animal models, raising scientific and ethical issues.
In our NC3R Project Grant, we developed a protocol for the preparation and ex vivo culture of superficial cardiac slices, comprising of the epicardial layer and the underlying myocardial tissue (EpCardio-TS), from abattoir porcine hearts (10-20 slices/heart) without further burden to animal life. Thanks to their structural complexity, representative of the heart tissue, and the high number slices obtained from each heart, this model allows the high throughput approach and the batch consistency needed for gene/drug discovery. Using a nanomaterial tool (nanoneedles) and a decolouration protocol (CUBIC), epicardial cells in EpCardio-TS can be transfected and/or labelled locally and visualised through the thickness of the slice.
This project aims at deploying our EpCardio-TS to two end user labs: Prof Stevens' lab at Imperial College London (primary end-user, London, UK) and Dr Martinez-Estrada's lab at University of Barcelona (secondary end-user, Barcelona, Spain).
Both end-user labs have ongoing projects aimed at the discovery of new genes influencing the epicardial reparative function and are planning to test the candidate genes in animal models of myocardial infarction. The introduction of EpCardio-TS to these labs will replace partially their reliance on small animals to screen their gene therapy candidates by an estimated 20%. The testimony and publications arising from the work of these outstanding collaborators, together with our planned local and international presentations, will further support the visibility and dissemination of EpCardio-TS.
In our NC3R Project Grant, we developed a protocol for the preparation and ex vivo culture of superficial cardiac slices, comprising of the epicardial layer and the underlying myocardial tissue (EpCardio-TS), from abattoir porcine hearts (10-20 slices/heart) without further burden to animal life. Thanks to their structural complexity, representative of the heart tissue, and the high number slices obtained from each heart, this model allows the high throughput approach and the batch consistency needed for gene/drug discovery. Using a nanomaterial tool (nanoneedles) and a decolouration protocol (CUBIC), epicardial cells in EpCardio-TS can be transfected and/or labelled locally and visualised through the thickness of the slice.
This project aims at deploying our EpCardio-TS to two end user labs: Prof Stevens' lab at Imperial College London (primary end-user, London, UK) and Dr Martinez-Estrada's lab at University of Barcelona (secondary end-user, Barcelona, Spain).
Both end-user labs have ongoing projects aimed at the discovery of new genes influencing the epicardial reparative function and are planning to test the candidate genes in animal models of myocardial infarction. The introduction of EpCardio-TS to these labs will replace partially their reliance on small animals to screen their gene therapy candidates by an estimated 20%. The testimony and publications arising from the work of these outstanding collaborators, together with our planned local and international presentations, will further support the visibility and dissemination of EpCardio-TS.
Planned Impact
EpCardio-TS is an ex vivo model for the study of the epicardial cell reparative potential and the development of targeted therapeutic approaches. We identified 2 labs that are currently using small animal models that could be replaced by EpCardio-TS and will provide extensive training and support to these end-users in order to reduce the number of animals used locally.
Our estimates of the number of small animals replaceable by EpCardio-TS are based on our previous experience and the detailed information provided by the end-users. Prof Stevens was planning study 5-10 genes in vivo, in a rat model of myocardial infarction. Since a previous study on a single gene required performing myocardial infarction on 40 rats, we can expect the use of 200-400 rats. Dr Martinez-Estrada aims at investigating 5-10 genes previously identified during development, on the adult epicardium by performing myocardial infarction in epicardial-specific KO mice. These experiments require long breading schemes (at least 50 mice/gene), followed by the induction of myocardial infarction in 50 mice/gene. To study 5-10 genes, we can estimate the use of 500-1000 animals for breeding and the myocardial infarction model.
This grant will ensure the adoption of EpCardio-TS in those two labs, where it will help identifying ex vivo the most promising genes, before animal experimentation. Assuming conservatively that the ex vivo study can exclude 1 in 5 genes prior to the in vivo phase, we can calculate a replacement of at least 20% of the animals, amounting to 140-280 animals not undergoing regulated procedures.
These calculations do not include the potential for further animal replacement granted by the engagement of new collaborators, including but not limited to, AstraZeneca and the University of Oxford that have already expressed an interest.
The strategy we propose to achieve our replacement target includes the following steps: (1) extensive training and support to the end-users to transfer EpCardio-TS to their labs; (2) in situ practical support to identify adequate equipment and tissue sources; (3) engagement of other interested laboratories at end-user institutions through invited presentations; (4) wider promotion of the model by production of method papers, website contents and posters/presentations; (5) increased visibility to other fields of science due to end-users presentations and papers; (6) developer presentations to engage prospective new users such as AstraZeneca and University of Oxford
We estimate that our extensive dissemination plan will help ensure the engagement of the laboratories that have already expressed interest in EpCardio-TS. The further adoption of our model in laboratories such as Prof Emanueli's laboratory in Imperial College London, Dr Smart's laboratory at Oxford University or AstraZeneca will create additional impact to EpCardio-TS in the replacement/reduction of animal use and its scientific visibility and attractiveness.
We envision EpCardio-TS having an economic impact by reducing the cost of the projects by limiting the animal expenditure, therefore benefitting the research groups and the funders. We also expect the translatability of the results obtained using EpCardio-TS to human subjects to be superior, as compared to small animal studies. This is an extremely relevant issue in the pharmaceutical industry, as shown by AstraZeneca's support letter.
Our system is based on commonly available cell culture and imaging facilities; we also established that both end users have access to vibratomes to cut the slices. The training provided and the online resources available will reduce any technical challenge.
The nanoneedles are available at Imperial College London and can be provided freely through collaborations with King's College London, subject to Material Transfer Agreement.
Our estimates of the number of small animals replaceable by EpCardio-TS are based on our previous experience and the detailed information provided by the end-users. Prof Stevens was planning study 5-10 genes in vivo, in a rat model of myocardial infarction. Since a previous study on a single gene required performing myocardial infarction on 40 rats, we can expect the use of 200-400 rats. Dr Martinez-Estrada aims at investigating 5-10 genes previously identified during development, on the adult epicardium by performing myocardial infarction in epicardial-specific KO mice. These experiments require long breading schemes (at least 50 mice/gene), followed by the induction of myocardial infarction in 50 mice/gene. To study 5-10 genes, we can estimate the use of 500-1000 animals for breeding and the myocardial infarction model.
This grant will ensure the adoption of EpCardio-TS in those two labs, where it will help identifying ex vivo the most promising genes, before animal experimentation. Assuming conservatively that the ex vivo study can exclude 1 in 5 genes prior to the in vivo phase, we can calculate a replacement of at least 20% of the animals, amounting to 140-280 animals not undergoing regulated procedures.
These calculations do not include the potential for further animal replacement granted by the engagement of new collaborators, including but not limited to, AstraZeneca and the University of Oxford that have already expressed an interest.
The strategy we propose to achieve our replacement target includes the following steps: (1) extensive training and support to the end-users to transfer EpCardio-TS to their labs; (2) in situ practical support to identify adequate equipment and tissue sources; (3) engagement of other interested laboratories at end-user institutions through invited presentations; (4) wider promotion of the model by production of method papers, website contents and posters/presentations; (5) increased visibility to other fields of science due to end-users presentations and papers; (6) developer presentations to engage prospective new users such as AstraZeneca and University of Oxford
We estimate that our extensive dissemination plan will help ensure the engagement of the laboratories that have already expressed interest in EpCardio-TS. The further adoption of our model in laboratories such as Prof Emanueli's laboratory in Imperial College London, Dr Smart's laboratory at Oxford University or AstraZeneca will create additional impact to EpCardio-TS in the replacement/reduction of animal use and its scientific visibility and attractiveness.
We envision EpCardio-TS having an economic impact by reducing the cost of the projects by limiting the animal expenditure, therefore benefitting the research groups and the funders. We also expect the translatability of the results obtained using EpCardio-TS to human subjects to be superior, as compared to small animal studies. This is an extremely relevant issue in the pharmaceutical industry, as shown by AstraZeneca's support letter.
Our system is based on commonly available cell culture and imaging facilities; we also established that both end users have access to vibratomes to cut the slices. The training provided and the online resources available will reduce any technical challenge.
The nanoneedles are available at Imperial College London and can be provided freely through collaborations with King's College London, subject to Material Transfer Agreement.
